Cloning Of Carotenoid Biosynthesis Enzyme Genes From Endophytic Fungi Of Crocus Sativus L. And Optimal Culture Of Recombinant

Crocus sativus L.is a very precious traditional Chinese medicine.However,the yield is limited and the price is expensive.Endophytic fungi of medicinal plants can produce metabolites with medicinal value.In this paper,carotenoid metabolic pathway synthase genes,including Cs Crt Y(lycopene cyclase gene),Cs ZCD(zeaxanthin lyase gene)and Cs ALDH(aldehyde dehydrogenase)gene family of Cs ALDH2/3/5,were cloned from Crocus sativus L.endophytic fungus 3P1CQ1 and the physicochemical properties and structure of related proteins were predicted and analyzed.The related proteins were analyzed by bioinformatics,including open reading frame analysis,physical and chemical property analysis,transmembrane domain prediction,secondary and tertiary structure prediction.This thesis laid a foundation for the study of carotenoid production by endophytic fungi.In recent years,the report of transferring heterologous genes into E.coli to synthesize carotenoids has been widely concerned.In this paper,the culture conditions of E.coli recombinant containing carotenoid biosynthesis gene of Crocus sativus L.had been optimized.Single factor temperature,culture medium,carbon source,inorganic nitrogen source and p H,were screened.On this basis,the results of orthogonal experiment showed that the best combination for the growth of this strain was glucose 5g/L,sodium nitrate 5g/L,p H 5.5 ±0.05,inoculation quantity 15%.The growth dynamic process of the orthogonal optimization results was investigated,and the expression was induced in the late logarithmic growth stage of 40 h culture.By HPLC analysis of metabolites,four carotenoids with retention time of 22.8 min,25.7min,33.5 min and 35.1min were obtained,which were 20.9,38.4,2.7and 23.1 times higher than those of the control group,respectively.